Osteoblasts make bone, a dense extracellular matrix of mainly type I collagen and hydroxyapatite mineral in an isolated compartment. Mineral deposition by phosphate production yields acid. Thus, osteoblasts must remove the acid created by mineral deposition. Our preliminary data include direct demonstration that matrix pH inside the osteoblast epithelium varies independently of extracellular pH in bone. Our work supports strongly the premise that osteoblasts alkalinize the bone matrix, although gaps in understanding persist. Many aspects of collagen secretion, phosphate production, and calcium transport are well studied, but proton transport across osteoblast epithelium is studied minimally other than in our work. We will use innovative methods, membrane transport, live cell imaging of organ explants, and surface plasmon resonance, to study the pH and mineralization of bone matrix. We expect to characterize components of the osteoblast proton transport in detail, and to define clearly the nature of mineral deposition on the bone collagen matrix. As we develop the molecular basis for these transport pathways we expect that molecular targets for therapeutic intervention will become available to manipulate bone mineralization in vitro and in vivo. Aim 1. Regulation of acid transport in active and inactive osteoblasts will directly address the hypothesis that acid transport is required to maintain bone mineral, and that much higher transport activity is regulated to allow bone mineralization to occur. We will study this by isolating active and inactive osteoblasts from rabbit spine separating activie and inactive by size. We will measure the amount and activity of acid transporting membrane proteins, as well as regulatory proteins for the acid transport process. In addition, we will produce osteoblasts in vitro, following bone formation, isolating transport proteins from cells as a function of activity. This will be done in normal cells and osteoclasts without and with over-expression of NHE1, ClC-3 or both. It is expected that bone formation and activity will be stimulated by over-expression of these transport proteins. Aim 2. Fluorescent visualization of live cell osteoblast proton fluxes will directly test the hypothesis that vectorial transport of protons across the osteoblast epithelium establishes a pH gradient with extracellular pH alkalinization due to the activity of the Cl/H exchanger ClC-3 at the basolateral membrane. Osteoblast secreted matrix calcium and pH sensors with enable spatiotemporal detection of mineral and proton fluxes. Aim 3. Parameters that affect mineral deposition on type I collagen will be determined using surface plasmon resonance. We will use collagens that do (Type I) or do not normally mineralize biologically (Type II) to explore the influence of collagen structure and the effects of osteopontin, osteocalcin and others to introduce regulatory influences. Time, H+, Ca2+ and phosphate will be primary independent variables. We focus on novel mechanisms supporting formation of mineralized bone matrix, specifically acid transport. These are important poorly studied elements of bone formation, and also potential novel therapeutic targets.